Analysis of the periplasmic proteome of <b><i>Pseudomonas aeruginosa</i></b>, a metabolically versatile opportunistic pathogen

Imperi, Francesco; Ciccosanti, Fabiola; Perdomo, Ariel Basulto; Tiburzi, Federica; Mancone, Carmine; Alonzi, Tonino; Ascenzi, Paolo; Piacentini, Mauro; Visca, Paolo; Fimia, Gian María

doi:10.1002/pmic.200800618

Cited by 85 publications

(74 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The current understanding of FliC function localizes the protein in the flagellum outside the cell, while the ATP- dependent DnaK chaperone is considered to be cytoplasmic. However, E. coli FliC and several other FliC proteins, including P. aeruginosa FliC, were detected in the inner membrane fraction and the periplasm during various proteomic analyses (38)(39)(40)(41)(42)(43). In multiple approaches, the FliC protein was detected together with NirS (41) or even with NirS and DnaK (43).…”

Section: Discussionmentioning

confidence: 99%

A Periplasmic Complex of the Nitrite Reductase NirS, the Chaperone DnaK, and the Flagellum Protein FliC Is Essential for Flagellum Assembly and Motility in Pseudomonas aeruginosa

et al. 2015

View full text Add to dashboard Cite

Pseudomonas aeruginosa is a ubiquitously occurring environmental bacterium and opportunistic pathogen responsible for various acute and chronic infections. Obviously, anaerobic energy generation via denitrification contributes to its ecological success. To investigate the structural basis for the interconnection of the denitrification machinery to other essential cellular processes, we have sought to identify the protein interaction partners of the denitrification enzyme nitrite reductase NirS in the periplasm. We employed NirS as an affinity-purifiable bait to identify interacting proteins in vivo. Results obtained revealed that both the flagellar structural protein FliC and the protein chaperone DnaK form a complex with NirS in the periplasm. The interacting domains of NirS and FliC were tentatively identified. The NirS-interacting stretch of amino acids lies within its cytochrome c domain. Motility assays and ultrastructure analyses revealed that a nirS mutant was defective in the formation of flagella and correspondingly in swimming motility. In contrast, the fliC mutant revealed an intact denitrification pathway. However, deletion of the nirF gene, coding for a heme d 1 biosynthetic enzyme, which leads to catalytically inactive NirS, did not abolish swimming ability. This pointed to a structural function for the NirS protein. FliC and NirS were found colocalized with DnaK at the cell surface of P. aeruginosa. A function of the detected periplasmic NirS-DnaK-FliC complex in flagellum formation and motility was concluded and discussed. IMPORTANCEPhysiological functions in Gram-negative bacteria are connected with the cellular compartment of the periplasm and its membranes. Central enzymatic steps of anaerobic energy generation and the motility mediated by flagellar activity use these cellular structures in addition to multiple other processes. Almost nothing is known about the protein network functionally connecting these processes in the periplasm. Here, we demonstrate the existence of a ternary complex consisting of the denitrifying enzyme NirS, the chaperone DnaK, and the flagellar protein FliC in the periplasm of the pathogenic bacterium P. aeruginosa. The dependence of flagellum formation and motility on the presence of an intact NirS was shown, structurally connecting both cellular processes, which are important for biofilm formation and pathogenicity of the bacterium. P seudomonas aeruginosa is a metabolically versatile bacterium inhabiting multiple environmental niches (1). It is known for its highly efficient growth in the absence of oxygen. Fast anaerobic growth is mediated via the utilization of different N-oxides as electron acceptors in the respiratory chains of denitrification (2). Anaerobic respiratory growth via denitrification also sustains biofilm formation on environmental surfaces, in the mucus in the lung of cystic fibrosis patients, on the epithelium of the urinary tract infected individuals, and in burn wounds (3).The periplasm, the cellular compartment bounded by the cytoplasmi...

show abstract

Section: Discussionmentioning

confidence: 99%

A Periplasmic Complex of the Nitrite Reductase NirS, the Chaperone DnaK, and the Flagellum Protein FliC Is Essential for Flagellum Assembly and Motility in Pseudomonas aeruginosa

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Periplasmic fractions were obtained by spheroplasting P. aeruginosa cells with lysozyme and MgCl 2 as described (30). Briefly, bacterial pellets (5 ϫ 10 9 cells) were suspended in 1 ml of 10 mM Tris-HCl (pH 8.4), 200 mM MgCl2, 0.5 mg/ml lysozyme, and incubated for 30 min at room temperature with gentle shaking.…”

Section: Construction Of Deletion Mutants and Complementingmentioning

confidence: 99%

“…Cell debris were removed to obtain the spheroplasts fractions. The specificity and purity of cell fractions were verified by using specific subcellular protein markers as described (30). PVD Measurements.…”

Section: Construction Of Deletion Mutants and Complementingmentioning

confidence: 99%

Molecular basis of pyoverdine siderophore recycling in Pseudomonas aeruginosa

Imperi

Tiburzi

Visca

2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

200

219

View full text Add to dashboard Cite

The siderophore pyoverdine (PVD) is a primary virulence factor of the human pathogenic bacterium Pseudomonas aeruginosa, acting as both an iron carrier and a virulence-related signal molecule. By exploring a number of P. aeruginosa candidate systems for PVD secretion, we identified a tripartite ATP-binding cassette efflux transporter, here named PvdRT-OpmQ, which translocates PVD from the periplasmic space to the extracellular milieu. We show this system to be responsible for recycling of PVD upon internalization by the cognate outer-membrane receptor FpvA, thus making PVD virtually available for new cycles of iron uptake. Our data exclude the involvement of PvdRT-OpmQ in secretion of de novo synthesized PVD, indicating alternative pathways for PVD export and recycling. The PvdRT-OpmQ transporter is one of the few secretion systems for which substrate recognition and extrusion occur in the periplasm. Homologs of the PvdRT-OpmQ system are present in genomes of all fluorescent pseudomonads sequenced so far, suggesting that PVD recycling represents a general energysaving strategy adopted by natural Pseudomonas populations.ATP-binding-cassette transporter ͉ periplasm ͉ iron ͉ fluorescent Pseudomonas ͉ secretion

show abstract

“…However, ectopic expression of muiA has been shown to suppress alginate overproduction in mucoid strains that retain WT MucA (52). Neither MuiA nor PA4495 has any significant homology to any previously characterized proteins, but both proteins are predicted to contain N-terminal secretion signals and have been found experimentally in the periplasm (53). To test whether MuiA or PA4495 contributed to the inhibition of swarming motility exhibited by the ⌬sbrR mutant strain, we generated PA14 ⌬sbrR ⌬muiA double mutants and PA14 ⌬sbrR ⌬PA4495 double mutants.…”

Section: Fig 2 Sbrr Is An Anti-factor That Directly Interacts With Sbmentioning

confidence: 99%

σ Factor and Anti-σ Factor That Control Swarming Motility and Biofilm Formation in Pseudomonas aeruginosa

2016

View full text Add to dashboard Cite

Pseudomonas aeruginosa is capable of causing a variety of acute and chronic infections. Here, we provide evidence that sbrR (PA2895), a gene previously identified as required during chronic P. aeruginosa respiratory infection, encodes an anti-factor that inhibits the activity of its cognate extracytoplasmic-function factor, SbrI (PA2896). Bacterial two-hybrid analysis identified an N-terminal region of SbrR that interacts directly with SbrI and that was sufficient for inhibition of SbrI-dependent gene expression. We show that SbrI associates with RNA polymerase in vivo and identify the SbrIR regulon. In cells lacking SbrR, the SbrI-dependent expression of muiA was found to inhibit swarming motility and promote biofilm formation. Our findings reveal SbrR and SbrI as a novel set of regulators of swarming motility and biofilm formation in P. aeruginosa that mediate their effects through muiA, a gene not previously known to influence surface-associated behaviors in this organism. IMPORTANCEThis study characterizes a factor/anti-factor system that reciprocally regulates the surface-associated behaviors of swarming motility and biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. We present evidence that SbrR is an anti-factor specific for its cognate factor, SbrI, and identify the SbrIR regulon in P. aeruginosa. We find that cells lacking SbrR are severely defective in swarming motility and exhibit enhanced biofilm formation. Moreover, we identify muiA (PA1494) as the SbrI-dependent gene responsible for mediating these effects. SbrIR have been implicated in virulence and in responding to antimicrobial and cell envelope stress. SbrIR may therefore represent a stress response system that influences the surface behaviors of P. aeruginosa during infection.T he Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen notorious for being the principal cause of morbidity and mortality in cystic fibrosis (CF) patients (1). In patients with CF, chronic pulmonary colonization by P. aeruginosa leads to chronic inflammation, progressive loss of lung function, and eventually respiratory failure and death (1). P. aeruginosa is also the fifth leading cause of nosocomial infections overall in the United States and is the second most common cause of ventilator-associated pneumonia (VAP) and catheter-associated urinary tract infections (CAUTI) (2, 3). In patients with VAP or CAUTI, P. aeruginosa grows as a biofilm on endotracheal tubes and catheters, respectively (4-6). In addition, P. aeruginosa is thought to persist as a biofilm in the CF lung (7). P. aeruginosa biofilms are associated with chronic infection and exhibit increased antibiotic resistance and resistance to clearance by the immune system (8). Thus, the ability to form biofilms contributes significantly to the clinical burden of P. aeruginosa infection.In P. aeruginosa, growth as a biofilm is inversely regulated with a cooperative form of multicellular surface motility called swarming (9-12). Swarming motility is flagell...

show abstract

Analysis of the periplasmic proteome of Pseudomonas aeruginosa, a metabolically versatile opportunistic pathogen

Cited by 85 publications

References 43 publications

A Periplasmic Complex of the Nitrite Reductase NirS, the Chaperone DnaK, and the Flagellum Protein FliC Is Essential for Flagellum Assembly and Motility in Pseudomonas aeruginosa

A Periplasmic Complex of the Nitrite Reductase NirS, the Chaperone DnaK, and the Flagellum Protein FliC Is Essential for Flagellum Assembly and Motility in Pseudomonas aeruginosa

Molecular basis of pyoverdine siderophore recycling in Pseudomonas aeruginosa

σ Factor and Anti-σ Factor That Control Swarming Motility and Biofilm Formation in Pseudomonas aeruginosa

Contact Info

Product

Resources

About